Cyclic AMP binds to its receptor protein CRP, and induces a conformational change (allosteric transition) in the protein. Cyclic AMP.CRP complex, and not free CRP, binds at or near many promoters of E. coli in a sequence-specific way and either represses or activates transcription. We have isolated and characterized several classes of mutations in the crp gene, which identify the amino acids participating in the cyclic AMP-induced allosteric shift in the protein. On the basis of these results and the available three-dimensional structure of the protein, we proposed that cyclic AMP brings about: (1) a hinge reorientation to eject the DNA-binding F c-helices; (2) proper alignment between two subunits; (3) an adjustment between the position of the two domains. Extensive amino acid substitution at some of these positions further supports the idea that hinge movement involving interhelical polar-polar interactions and/or Van der Waals repulsion are an essential part of the activation of CRP. We have further shown, by site-directed mutagenesis, that a hydrogen bond formation between amino acids 171 and 63 participates in domain- domain alignment for CRP activation. Using a DNA bending vector developed in this laboratory, we have found that CRP binding induces DNA bending at all tested promoters with a CRP binding site whether the promoters are activable or repressible by CRP. The following two findings strongly support the idea that CRP activates RNA polymerase by a direct protein-protein contact and not by sending a signal through DNA: (1) We have found that substantially increasing the distance between the RNA polymerase binding site and the CRP binding site in lacP does not affect CRP activation of lacP. (2) Additionally, we have clearly demonstrated, using one of our CRP mutants, that CRP and RNA polymerase bind to DNA co- operatively.